The effects of physical activity on apoptosis and lubricin expression in articular cartilage in rats with glucocorticoid-induced osteoporosis.

Glucocorticoids are considered the most powerful anti-inflammatory and immunomodulating drugs. However, a number of side-effects are well documented in different diseases, including articular cartilage, where increases or decreases in the synthesis of hormone-dependent extracellular matrix components are seen. The objective of this study has been to test the effects of procedures or drugs affecting bone metabolism on articular cartilage in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity with treadmill and vibration platform training on articular cartilage. The animals were divided into 5 groups, and bone and cartilage evaluations were performed using whole-body scans and histomorphometric analysis. Lubricin and caspase-3 expression were evaluated by immunohistochemistry, Western blot analysis and biochemical analysis. These results confirm the beneficial effect of physical activity on the articular cartilage. The effects of drug therapy with glucocorticoids decrease the expression of lubricin and increase the expression of caspase-3 in the rats, while after physical activity the values return to normal compared to the control group. Our findings suggest that it might be possible that mechanical stimulation in the articular cartilage could induce the expression of lubricin, which is capable of inhibiting caspase-3 activity, preventing chondrocyte death. We can assume that the physiologic balance between lubricin and caspase-3 could maintain the integrity of cartilage. Therefore, in certain diseases such as osteoporosis, mechanical stimulation could be a possible therapeutic treatment. With our results we can propose the hypothesis that physical activity could also be used as a therapeutic treatment for cartilage disease such as osteoarthritis.

Expression and localization of aquaporin-1 in temporomandibular joint disc with internal derangement.

BACKGROUND: Internal derangement is the most frequent arthropathy affecting the temporomandibular joint, where its commonest form is anterior disc displacement with or without reduction. Despite the frequency of the disorder, the biochemical features of displaced discs are still unclear. METHODS: We investigated the expression pattern and localization of aquaporin-1, an important channel protein involved in plasma membrane water permeability, in patients with anterior disc displacement (both with and without reduction), with a view to assessing the characteristics of local tissue responses to the microenvironmental changes induced by abnormal mechanical loading of the displaced disc. Protein expression was studied by immunohistochemistry in different areas of discs from 18 patients with anterior disc displacement with or without reduction and in four normal controls. RESULTS: A greater proportion of cells immunopositive for aquaporin-1 were detected in diseased than in normal discs. Whereas protein expression was substantially similar in the different areas of normal discs, a significantly larger number of immunopositive cells were detected in the posterior band of displaced discs without reduction and in the anterior and intermediate bands of those with reduction. CONCLUSIONS: These findings suggest that aquaporin-1 is expressed and upregulated in temporomandibular joint with anterior disc displacement (both with and without reduction).

Expression of ß-defensin-4 in "an in vivo and ex vivo model" of human osteoarthritic knee meniscus.

PURPOSE: To investigate, for the first time, the expression of ß-defensins-4, by immunohistochemistry and western blotting, in OA meniscus versus control meniscus, thus providing new insights into the physiological processes of meniscus repairing. METHOD: ß-defensins-4 was studied in vivo, in knee osteoarthritic menisci obtained from 30 patients (20 men and 10 women) who underwent isolated arthroscopic partial medial or lateral meniscectomy, and in vitro on fibrochondrocyte cells from human OA knee menisci. The study was conducted using morphological, immunohistochemical, and Western blot analysis. RESULTS: The histological results demonstrated structural alterations and cracks of OA menisci accompanied by a very strong ß-defensin-4 immunohistochemical staining. The Western blot analysis confirmed also a strong expression of ß-defensin-4 in OA fibrochondrocyte cells. CONCLUSION: The present study suggests an activation of ß-defensin-4 induction, in human knee meniscus induced by the OA inflammatory process. It may represent an endogenous antibiotic defense mechanism accompanied by an intrinsic effort of tissue remodeling in OA articular joints. In conclusion, the present paper suggests the clinical relevance of ß-defensin-4 in the prospective of future alternative medical treatment for OA.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5) in Peyronie's disease. A biomolecular study of apoptosis activation.

INTRODUCTION: Peyronie's disease (PD) is a connective tissue disorder of tunica albuginea (TA), a thick fibrous sheath surrounding the corpora cavernosa of the penis. Relatively, little is known about the disease itself. AIM: To investigate whether the apoptosis cascade in degenerated and macroscopically deformed TA from men with PD is activated through the extrinsic pathway, by assessing the immunoexpression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor, DR5. METHODS: TA plaques from 15 men with PD and from four unaffected men were processed for TRAIL and DR5 immunohistochemistry and Western blot analysis. MAIN OUTCOME MEASURES: A greater understanding of the pathophysiology of PD through a molecular approach, to gain insights that may lead to novel forms of treatment. RESULTS: Activation of the apoptosis mechanisms through the extrinsic pathway was demonstrated by TRAIL and DR5 overexpression in fibroblasts and myofibroblasts from affected TA. CONCLUSION: The finding that apoptosis activation in TA plaques occurs, at least in part, via the extrinsic pathway may help devise novel therapeutic options for these patients.

Characterization of apoptosis in articular cartilage derived from the knee joints of patients with osteoarthritis.

PURPOSE: the present study was conduced in order to analyse the molecular changes during the apoptotic cascade in knee articular cartilage of patients with OA. METHOD: articular cartilage specimens were assessed by histology (Haematoxylin and Eosin), histochemistry (Masson's Trichromic and Alcian Blue), immunohistochemistry through TRAIL, DR5 and Caspase-3, TUNEL and Hoechst staining in fresh isolated chondrocytes. RESULTS: histology results demonstrated the structural alterations in the articular knee cartilage with OA, and histochemistry results demonstrated the presence of matrix calcification and a proteoglycans reduction. Immunohistochemistry staining showed that structural alterations, matrix calcification and a proteoglycans reduction coincided with an increase in apoptotic cells when compared to normal cartilage; however, this cellular mechanism of death was demonstrated by TUNEL and Hoechst 33258 staining in fresh isolated chondrocytes. CONCLUSION: in this study, we demonstrated an apoptosis activation by the extrinsic pathway in OA cartilage. The apoptosis-positive cells might be due to a protection mechanism after sublethal injury, in particular, represented by an increased survival of chondrocytes that are able to participate in the repair process.

Early effects of aluminum chloride on beta-secretase mRNA expression in a neuronal model of beta-amyloid toxicity.

Amyloid beta peptide (Abeta), generated by proteolytic cleavage of the amyloid precursor protein (APP), plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). The key step in the generation of Abeta is cleavage of APP by beta-secretases (beta-site APP-cleaving enzyme 1 (BACE1) and BACE2). There has been suggestion of interaction between aluminum and several AD-associated pathways. However, the underlying mechanisms still remain unclear. Here, we report the effects of aluminum chloride (AlCl(3)) in Abeta-induced toxicity using differentiated neuronal SH-SY5Y cells. The metal significantly enhances Abeta-induced cell death at concentrations ranging from 50 to 300 microM after 24 and 48 h. After 72 and 96 h treatment, cell death is increased already at 10 microM. Early coexposure of cells to 10 microM AlCl(3) and 2 microM Abeta differentially affected beta-secretase mRNA levels as compared to single Abeta treatment after 1 and 3 h. BACE1 levels were slightly reduced after 1 h and significantly increased after 3 h exposure, whereas BACE2 levels were increased at both times considered. Both genes' mRNA levels were downregulated at longer times (6, 12, and 24 h). Although these results indicate that aluminum toxicity is correlated to changes in both BACE1 and BACE2 expression levels, the subsequent common downregulation observed suggests that aluminum involvement in the Abeta cascade is subtle, and other underlying mechanisms might be involved.

Mineral fiber-mediated activation of phosphoinositide-specific phospholipase c in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells.

Given the role of phosphoinositide-specific phospholipase C (PLC) isozymes in the control of cell growth and differentiation we were prompted to analyze the expression of some of these PLC in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells. The effects of several fluoro-edenite fibers were compared with those of tremolite, a member of the calcic amphibole group of asbestos that originates from Calabria (Italy), and crocidolite, that, due to its high toxicity, is one of the most studied asbestos amphiboles. Our data show an increased expression of both PLC beta1 and PLC gamma1 in A549 cells treated with asbestos-like fibers, hinting at a role of PLC signalling in those cancerous cells.

Parkin expression profile in dopamine d3 receptor knock-out mice brains.

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms' distribution pattern and in exons' expression both in wild type controls and dopamine D3 receptor's knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.

Immunohistochemical changes in vulnerable rat brain regions after reversible global brain ischaemia.

Human global ischaemia was simulated in adult rats by inducing 20 min brain ischaemia and 60 min post-ischaemic recirculation. Immunohistochemical expression of MMP-9, TIMP-3, Bax and Bcl-2, and DNA fragmentation (with the TUNEL reaction) were investigated. The morphological data showed different neuronal responses in the hippocampus compared with the cerebral and cerebellar cortices. MMP-9 immunoreactivity was different in the hippocampus, particularly in dentate gyrus and the CA1 region, compared with these cortices. Negative TIMP-3 staining in ischaemic hippocampal neurons may indicate a loss of its inhibitory activity on MMP-9 that could enhance cell death. Bcl-2 down regulation, Bax positivity and TUNEL+ type II cells in the dentate gyrus granular layer could be responsible for induction of apoptotic death in CA1 hippocampal pyramidal cells via loss of fibre input. Results suggest differential behaviours of neural cells after 60 min reperfusion.

Effect of adrenomedullin on c-Met receptor expression after reserpine-induced gastric damage in the rat.

Here, we show an increase in c-Met receptor expression during reserpine-induced gastric damage in the rat, as assessed by immunohistochemistry. Pretreatment of animals with adrenomedullin prevented this increase in c-Met expression. c-Met immunoreactivity was localized in gastric glands. c-Met immunoreactivity was associated with increased phosphorylation of c-Met receptor and extracellular signal-regulated kinase (ERK(1/2)). Our results suggest that both adrenomedullin and c-Met act as parallel defence mechanisms during pharmacologically induced gastric mucosa injury.

Acute injury affects lubricin expression in knee menisci: an immunohistochemical study.

The aim of this study was to investigate for the first time lubricin expression in intact menisci and in menisci from patients with recent knee joint injury using histology, immunohistochemistry, Western blotting and gene expression analysis, to provide insights into pathological processes affecting meniscal tissue. Lubricin expression was studied in vivo in 20 patients (14 males and 6 females) with recent joint injury subjected to arthroscopic partial meniscectomy and in vitro in fibroblast-like cells from meniscus tissue to establish whether it is down-regulated following acute traumatic knee injury. The control group consisted of cadaver donors with normal menisci. Histology demonstrated a normal tissue without structural changes in control samples and structural alterations and clefts in injured menisci. Very strong lubricin immunohistochemical staining was observed in intact menisci; in contrast weak staining was seen in injured menisci. Western blot and mRNA expression analysis also demonstrated strong lubricin expression in control cells and a negligible amount of lubricin in injured fibroblast-like cells. Our data provide information concerning the immediate in vivo response to injury of human knee menisci by documenting early changes in the boundary-lubricating ability of synovial fluid and articular cartilage integrity. These findings may provide the biological basis for developing novel medical therapies to be applied before surgical treatment to preserve tissue function and prevent cartilage damage.

Ameliorative effect of PACAP and VIP against increased permeability in a model of outer blood retinal barrier dysfunction.

Breakdown of outer blood retinal barrier (BRB) due to the disruption of tight junctions (TJs) is one of the main factors accounting for diabetic macular edema (DME), a major complication of diabetic retinopathy. Previously it has been shown that PACAP and VIP are protective against several types of retinal injuries. However, their involvement in the maintenance of outer BRB function during DME remains uncovered. Here, using an in vitro model of DME, we explored the effects of both PACAP and VIP. Human retinal pigment epithelial cells (ARPE19) were cultured for 26 days either in normal glucose (5.5 mM, NG) or in high glucose (25 mM, HG). In addition, to mimic the inflammatory aspect of the diabetic milieu, cells were also treated with IL-1ß (NG+IL-1ß and HG+IL-1ß). Effects of PACAP or VIP on cells permeability were evaluated by measuring both apical-to-basolateral movements of fluorescein isothyocyanate (FITC) dextran and transepithelial electrical resistance (TEER). Expression of TJ-related proteins was evaluated by immunoblot. Results demonstrated that NG+IL-1ß and, to a greater extent, HG+IL-1ß significantly increased FITC-dextran diffusion, paralleled by decreased TEER. PACAP or VIP reversed both of these effects. Furthermore, HG+IL-1ß-induced reduction of claudin-1 and ZO-1 expression was reversed by PACAP and VIP. Occludin expression was not affected in any of the conditions tested. Altogether, these finding show that both peptides counteract HG+IL-1ß-induced damage in ARPE19 cells, suggesting that they might be relevant to the maintenance of outer BRB function in DME.

ß-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold.

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate ß-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of ß-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong ß-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak ß-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of ß-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using ß-defensin-4 as a relevant marker.

Immunohistochemical expression of retinoblastoma and phospho-retinoblastoma protein in sheep lung exposed to fluoro-edenite fibers.

The immunohistochemical expression of retinoblastoma protein (Rb) and its phosphorylated form (pRb) was investigated in an in vivo sheep model, i.e., lung tissue exposed to asbestos-like fibers. Rb is a phosphoprotein with a central role in controlling the cell cycle, cell growth regulation, differentiation, and apoptosis. pRb functions as a checkpoint in the G(1) phase, favoring cell growth, while the hypophosphorylated form (Rb) not only promotes cell-cycle arrest and a return to the G(1) phase, but also appears to be a key player in most instances of apoptosis. pRb overexpression was detected in alveolar epithelium and the interstitium, especially close to the fluoro-edenite fibers, whereas Rb immunostaining was faint or absent. The data suggest that the altered balance between pRb and Rb expression may be a programmed response to protect the organism against uncontrolled cell proliferation.

PACAP and VIP affect NF1 expression in rat malignant peripheral nerve sheath tumor (MPNST) cells.

In our previous study we have identified PACAP, VIP and their receptors in rat malignant peripheral nerve sheath tumor (MPNST) cells, thus showing anti-apoptotic roles. Recently it has been shown that the tumor suppressor neurofibromin, encoded by the Neurofibromatosis type I (NF1) gene, promotes MPNST cells sensitivity to apoptosis after serum withdrawal. In the present study we investigated whether PACAP or VIP negatively regulate NF1 expression under normal or serum-dependent pro-apoptotic culture conditions. Results indicated that serum itself significantly influenced gene and protein levels. In fact, the low NF1 levels of cells cultured in normal serum-containing medium were remarkably increased in cells switched to low- or no-serum after 24h and 48 h. Treatment with 100 nM PACAP or VIP did not affect NF1 expression when using normal amounts of serum, whereas it significantly inhibited transcript and protein levels both in low- or no-serum cultured cells. In particular, PACAP reduced NF1 levels already after 24h in low-serum cultured cells, while VIP showed a similar effect only after serum deprivation. However, both PACAP and VIP downregulated gene and protein levels within 48 h either in low-dose and serum-starved cells. Results were confirmed by fluorescence microscopy, showing that 100 nM PACAP or VIP attenuated neurofibromin cytoplasmic localization only in low- or no-serum cultured cells. The present study provides a comprehensive analysis of both neuropeptides effect on NF1 expression in normal, low- or serum-starved MPNST cells, ameliorating the hypothesis that resistance to apoptosis in serum-deprived cells might be correlated to PACAP-/VIP-induced NF1 inhibition.

PACAP and VIP prevent apoptosis in schwannoma cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.

Bitumen products alter bax, bcl-2 and cytokeratin expression: an in vivo study of chronically exposed road pavers.

BACKGROUND: The skin of road pavers is chronically exposed to bitumen fumes, a mixture of volatile compounds and particles, containing several carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. Bitumen fumes can alter cutaneous barrier integrity in various ways and induce skin diseases. The present study was devised to investigate the expression of apoptosis proteins (bax and bcl-2) and the cytokeratin pattern in skin specimens from road paving workers exposed to bitumen fumes using immunohistochemical techniques. METHODS: Skin forearm punch biopsies from 16 occupationally exposed workers and an unexposed control group were processed for immunohistochemistry using a broad-spectrum anti-cytokeratin antibody and monoclonal antibodies for bax and bcl-2 immunostaining. Urinary 1-hydroxypyrene (1-OHP) was also determined. RESULTS: Morphological specimen evaluation showed epidermal thinning of exposed skin and flattened dermal papillae. In sections from exposed subjects, grade 3 bax overexpression and cytokeratin immunoreaction was detected in all layers, while bcl-2 expression was downregulated (grade 1) and confined to the basal layer. There was a significant difference in 1-OHP values between road pavers and the control group (p < 0.05). CONCLUSION: Overexpression of the cytokeratin pattern and bax and underexpression of bcl-2 in chronically bitumen-exposed skin suggest that bitumen fumes induce activation of apoptosis as a defense mechanism.